We plan to study mouse 3T3 fibroblasts (preadipocytes) as they differentiate into adipocytes in vitro. Using these cells we have already established that (a) when preadipocytes differentiate they acquire an ACTH-responsive adenylate cyclase system not present in the undifferentiated cell and that sensitivity of the adipocyte adenylate cyclase to beta-adrenergic amines is enhanced 10-15-fold; (b) there are high affinity insulin binding sites on the preadipocyte whose numbers increase about 2-fold upon differentiation. On the other hand, sensitivity to insulin as measured by glucose uptake and conversion to CO2 and lipid is not present in the preadipocytes and appears dramatically upon cellular differentiation. To elucidate the nature of the adipocyte differentiation process and the role of insulin and other hormones in altering function and metabolism in these cells we will: (a) study changes in hormone sensitivity during development. By combining assays for both hormone receptors and early biochemical actions of the hormones we will determine whether the changes involve synthesis of new receptors and/or coupling of receptors to adenylate cyclase; (b) correlate the development of insulin sensitivity with changes in intracellular cyclic nucleotide metabolism, lipid metabolism, alterations in plasma membrane composition and function in order to assess how coupling between insulin binding and insulin action takes place; (c) study in detail the phenomenon of "down regulation" of hormone (e.g., insulin) receptors; (d) develop variant cell lines defective in either insulin sensitivity or cyclic nucleotide function in an effort to pinpoint the role of insulin and cyclic nucleotides in fat cell development and function; (e) study the biochemical basis for the triggering of adipocyte differentiation by insulin, glucocorticoids and cyclic nucleotide phosphodiesterase inhibitors. Recent evidence suggests that two new plasma membrane proteins are formed in response to chronic administration of insulin and/or glucose deprivation and that these proteins are involved in glucose transport. We plan to study the biosynthesis of these proteins, their insertion into the membrane. Their role in glucose transport will be investigated in oriented membrane vesicles.